title: Reciprocal interferences of TNF-α and Wnt1/β-catenin signaling axes shift bone marrow-derived stem cells towards osteoblast lineage after ethanol exposure.
abstract: OBJECTIVE
  We have reported in a separate study that alcohol exposure triggers activation of the TNF-α signaling pathway leading to an adverse shift of multipotential mesenchymal stem cells in bone marrow (BMSCs) away from osteogenesis towards adipogenesis. However, inhibition of TNF-α signaling only yielded moderate inhibition of adipogenesis. Here we showed that in addition to promoting the TNF-α signaling, alcohol also suppressed the Wnt1/β-catenin signaling pathway.
  
  
  METHODS
  We treated primary BMSCs from human subjects with alcohol for 24 days. We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin.
  
  
  RESULTS
  Wnt/β-catenin signaling was markedly activated in BMSCs treated with osteogenic inducers relative to the control cells, as indicated by the increased levels of nuclear β-catenin along with reduced levels of cytosolic β-catenin, as well as increased protein levels of Wnt1. Activation of Wnt/β-catenin signaling was significantly suppressed in BMSCs exposed to alcohol, which was reflected by downregulated expression of osteogenic marker genes Osf2/Cbfa1, osteopontin and osteocalcin, upregulated adipogenic marker PPARγ2 and aP2, and reduced number of calcifcation nodules. In contrast, activation of Wnt/β-catenin signaling by BIO favored osteogenesis even in the presence of alcohol. Simultaneous activation of Wnt1 by BIO and inhibition of TNF-α by 3,6'-dithiothalidomide produced synergetic suppression of ethanol-induced adipogenic lineage compared to interference with either of them alone.
  
  
  CONCLUSIONS
  This remarkable shift of BMSCs towards osteoblast lineage suggests the superiority of concordant and reciprocal interferences of the TNF-α and Wnt/β-catenin pathways for promoting osteogenesis.
authors.affiliation: Southern Medical University, 2010 MD TongHe, Guangzhou, China.
authors.forename: Yueping
authors.initials: Y
authors.lastname: Chen
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.forename: Qingshui
authors.initials: Q
authors.lastname: Yin
authors.forename: Hui
authors.initials: H
authors.lastname: Gao
authors.forename: Panfeng
authors.initials: P
authors.lastname: Dong
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Kang
mesh_terms: D050156:Adipogenesis / Q000187:drug effects; D001854:Bone Marrow Cells / Q000166:cytology; D019070:Cell Lineage / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D000431:Ethanol / Q000633:toxicity*; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D010006:Osteoblasts / Q000166:cytology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D051155:Wnt1 Protein / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; C493410:FABP4 protein, human; D050556:Fatty Acid-Binding Proteins; D006575:Heterocyclic Compounds, 3-Ring; D047495:PPAR gamma; D014409:Tumor Necrosis Factor-alpha; C495247:WNT1 protein, human; D051155:Wnt1 Protein; C544261:XAV939; D051176:beta Catenin; D015675:Osteocalcin; D053495:Osteopontin; D000431:Ethanol